The objective of my proposal is to understand the molecular mechanism of calcium-triggered synaptic exocytosis. I will use single-molecule approaches to investigate how Munc18-1, complexin, synaptotagmin, NSF, and ?-SNAP regulate synaptic SNARE assembly and disassembly. SNARE proteins assemble via multiple intermediates to drive fusion of neurotransmitter-containing vesicles with the presynaptic plasma membrane. Key regulatory proteins target these intermediates to activate and/or inhibit SNARE assembly, enabling synaptic exocytosis to be triggered by the calcium influx induced by an action potential. After fusion, NSF and ?-SNAP disassemble the fully assembled SNARE complexes to recycle SNARE proteins, which is required for frequent neuron communication. The precise, rapid SNARE assembly and disassembly is central to calcium-triggered synaptic exocytosis and underlies our thinking, memory, emotions, and actions. Malfunctions in this process cause various neurological disorders and neurodegenerative diseases. Yet, the mechanism underlying the regulated SNARE assembly and disassembly is poorly understood. We have developed a unique single-molecule manipulation approach based on high-resolution optical tweezers to investigate dynamic SNARE assembly and disassembly. We have identified several intermediates of SNARE assembly and characterized the energies, and kinetics associated with these intermediates as well as the fully assembled SNARE complex for the first time, paving a new road to study SNARE regulation. We will expand our single-molecule approach to address several key questions: (1) What is the essential role of Munc18-1 in SNARE assembly and disassembly? (2) Whether and how complexin and synaptotagmin activate and/or clamp SNARE assembly in a calcium-dependent manner? (3) How NSF and ?-SNAP recognize and disassemble only the fully assembled SNARE complexes, but not the partially assembled SNARE intermediates essential for membrane fusion? Our research will provide unique insights into the mechanisms of regulated SNARE assembly and disassembly, calcium-triggered exocytosis, and associated diseases.